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1.  mRNA for the EAAC1 Subtype of Glutamate Transporter is Present in Neuronal Dendrites In Vitro and Dramatically Increases In Vivo After a Seizure 
Neurochemistry international  2010;58(3):366-375.
The neuronal Na+-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200 μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.
doi:10.1016/j.neuint.2010.12.012
PMCID: PMC3040252  PMID: 21185901
glutamate transport; EAAC1; EAAT3; epilepsy; pilocarpine; seizure; mRNA targeting
2.  Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria 
PLoS ONE  2012;7(3):e34015.
Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.
doi:10.1371/journal.pone.0034015
PMCID: PMC3316532  PMID: 22479505
3.  Group I mGluR-Regulated Translation of the Neuronal Glutamate Transporter, Excitatory Amino Acid Carrier 1 (EAAC1) 
Journal of neurochemistry  2011;117(5):812-823.
Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In the present study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of GluR2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin (mTOR) or an inhibitor of extracellular signal-regulated kinase (ERK). These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE.
doi:10.1111/j.1471-4159.2011.07233.x
PMCID: PMC3088777  PMID: 21371038
glutamate transport; EAAC1; epilepsy; pilocarpine; seizure; mGluR; dendritic targeting
4.  Inhibition of GTRAP3-18 May Increase Neuroprotective Glutathione (GSH) Synthesis 
Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration.
doi:10.3390/ijms130912017
PMCID: PMC3472789  PMID: 23109897
glutathione; cysteine uptake; GTRAP3-18; EAAC1; neurodegeneration
5.  A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy 
The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.
PMCID: PMC2483507  PMID: 12151515
EAAC1; transport; antisense; GABA; metabolism; epilepsy
6.  Proteomic analyses of retina of excitatory amino acid carrier 1 deficient mice 
Proteome Science  2007;5:13.
Background
Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter found in neuronal tissues and is extensively expressed in the retina. EAAC1 plays a role in a variety of neural functions, but its biological functions in the retina has not been fully determined. The purpose of this study was to identify proteins regulated by EAAC1 in the retina of mice. To accomplish this, we used a proteomics-based approach to identify proteins that are up- or down-regulated in EAAC1-deficient (EAAC1-/-) mice.
Results
Proteomic analyses and two-dimensional gel electorphoresis were performed on the retina of EAAC1-/- mice, and the results were compared to that of wild type mice. The protein spots showing significant differences were selected for identification by mass spectrometric analyses. Thirteen proteins were differentially expressed; nine proteins were up-regulated and five proteins were down-regulated in EAAC1-/- retina. Functional clustering showed that identified proteins are involved in various cellular process, e.g. cell cycle, cell death, transport and metabolism.
Conclusion
We identified thirteen proteins whose expression is changed in EAAC-/- mice retinas. These proteins are known to regulate cell proliferation, death, transport, metabolism, cell organization and extracellular matrix.
doi:10.1186/1477-5956-5-13
PMCID: PMC2014740  PMID: 17711584
7.  Detergent-Insoluble EAAC1/EAAT3 Aberrantly Accumulates in Hippocampal Neurons of Alzheimer’s Disease Patients 
Disturbed glutamate homeostasis may contribute to the pathological processes involved in Alzheimer’s disease (AD). Once glutamate is released from synapses or from other intracellular sources, it is rapidly cleared by glutamate transporters. EAAC1 (also called EAAT3 or SLC1A1) is the primary glutamate transporter in forebrain neurons. In addition to transporting glutamate, EAAC1 plays other roles in regulating GABA synthesis, reducing oxidative stress in neurons, and is important in supporting neuron viability. Currently, little is known about EAAC1 in AD. To address whether EAAC1 is disturbed in AD, immunohistochemistry was performed on tissue from hippocampus and frontal cortex of AD and normal control subjects matched for age and gender. While EAAC1 immunostaining in cortex appeared comparable to controls, in the hippocampus, EAAC1 aberrantly accumulated in the cell bodies and proximal neuritic processes of CA2–CA3 pyramidal neurons in AD patients. Biochemical analyses showed that Triton X-100-insoluble EAAC1 was significantly increased in the hippocampus of AD patients compared to both controls and Parkinson’s disease patients. These findings suggest that aberrant glutamate transporter expression is associated with AD-related neuropathology and that intracellular accumulation of detergent-insoluble EAAC1 is a feature of the complex biochemical lesions in AD that include altered protein solubility.
doi:10.1111/j.1750-3639.2008.00186.x
PMCID: PMC2657182  PMID: 18624794
Glutamate Uptake; Glutamate toxicity; Synaptic dysfunction; protein aggregation; neurodegeneration; memantine; excitotoxicity
8.  Cooperation of the Conserved Aspartate 439 and Bound Amino Acid Substrate Is Important for High-Affinity Na+ Binding to the Glutamate Transporter EAAC1 
The Journal of General Physiology  2007;129(4):331-344.
The neuronal glutamate transporter EAAC1 contains several conserved acidic amino acids in its transmembrane domain, which are possibly important in catalyzing transport and/or binding of co/countertransported cations. Here, we have studied the effects of neutralization by site-directed mutagenesis of three of these amino acid side chains, glutamate 373, aspartate 439, and aspartate 454, on the functional properties of the transporter. Transport was analyzed by whole-cell current recording from EAAC1-expressing mammalian cells after applying jumps in voltage, substrate, or cation concentration. Neutralization mutations in positions 373 and 454, although eliminating steady-state glutamate transport, have little effect on the kinetics and thermodynamics of Na+ and glutamate binding, suggesting that these two positions do not constitute the sites of Na+ and glutamate association with EAAC1. In contrast, the D439N mutation resulted in an approximately 10-fold decrease of apparent affinity of the glutamate-bound transporter form for Na+, and an ∼2,000-fold reduction in the rate of Na+ binding, whereas the kinetics and thermodynamics of Na+ binding to the glutamate-free transporter were almost unchanged compared to EAAC1WT. Furthermore, the D439N mutation converted l-glutamate, THA, and PDC, which are activating substrates for the wild-type anion conductance, but not l-aspartate, into transient inhibitors of the EAAC1D439 anion conductance. Activation of the anion conductance by l-glutamate was biphasic, allowing us to directly analyze binding of two of the three cotransported Na+ ions as a function of time and [Na+]. The data can be explained with a model in which the D439N mutation results in a dramatic slowing of Na+ binding and a reduced affinity of the substrate-bound EAAC1 for Na+. We propose that the bound substrate controls the rate and the extent of Na+ interaction with the transporter, depending on the amino acid side chain in position 439.
doi:10.1085/jgp.200609678
PMCID: PMC2151618  PMID: 17389249
9.  Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons 
Biochimica et Biophysica Acta  2013;1833(6):1421-1433.
RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [3H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca2 + oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2 + dynamics the in neurons.
Highlights
•The reticulon protein RTN1A is an ER-resident protein of unknown function.•We identified the intracellular calcium release channel ryanodine receptor RyR2 as a specific binding partner of RTN1A.•RNT1A co-immunoprecipitates and colocalizes with the ryanodine receptor RyR2 in neurons via its N-terminal domain.•Binding of RTN1A modulates the activity of RyR2.•RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2 + dynamics
doi:10.1016/j.bbamcr.2013.02.012
PMCID: PMC3636420  PMID: 23454728
RTN, reticulon protein; ER, endoplasmic reticulum; RyR, ryanodine receptor; RHD, reticulon homology domain; CICR, calcium induced calcium release; IPTG, isopropyl-β-d-thiogalactopyranoside; PB, phosphate buffer; DAB, 3,3′-diaminobenzidine tetrahydrochloride dihydrate; CA, cornu ammonis; Reticulon; Brain; Protein–protein interaction; Calcium homeostasis
10.  The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS 
The Journal of Neuroscience  2012;32(17):6000-6013.
The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm−3), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-d-aspartate (4-methoxy-7-nitroindolinyl-d-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and d-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.
doi:10.1523/JNEUROSCI.5347-11.2012
PMCID: PMC4031369  PMID: 22539860
11.  Early Intermediates in the Transport Cycle of the Neuronal Excitatory Amino Acid Carrier Eaac1 
The Journal of General Physiology  2001;117(6):547-562.
Electrogenic glutamate transport by the excitatory amino acid carrier 1 (EAAC1) is associated with multiple charge movements across the membrane that take place on time scales ranging from microseconds to milliseconds. The molecular nature of these charge movements is poorly understood at present and, therefore, was studied in this report in detail by using the technique of laser-pulse photolysis of caged glutamate providing a 100-μs time resolution. In the inward transport mode, the deactivation of the transient component of the glutamate-induced coupled transport current exhibits two exponential components. Similar results were obtained when restricting EAAC1 to Na+ translocation steps by removing potassium, thus, demonstrating (1) that substrate translocation of EAAC1 is coupled to inward movement of positive charge and, therefore, electrogenic; and (2) the existence of at least two distinct intermediates in the Na+-binding and glutamate translocation limb of the EAAC1 transport cycle. Together with the determination of the sodium ion concentration and voltage dependence of the two-exponential charge movement and of the steady-state EAAC1 properties, we developed a kinetic model that is based on sequential binding of Na+ and glutamate to their extracellular binding sites on EAAC1 explaining our results. In this model, at least one Na+ ion and thereafter glutamate rapidly bind to the transporter initiating a slower, electroneutral structural change that makes EAAC1 competent for further, voltage-dependent binding of additional sodium ion(s). Once the fully loaded EAAC1 complex is formed, it can undergo a much slower, electrogenic translocation reaction to expose the substrate and ion binding sites to the cytoplasm.
PMCID: PMC2232401  PMID: 11382805
glutamate transporter; charge movement; patch clamp; caged compounds; rapid kinetics
12.  EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone 
BMC Biochemistry  2006;7:10.
Background
Prostate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells.
Results
The studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells.
Conclusion
This study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.
doi:10.1186/1471-2091-7-10
PMCID: PMC1456973  PMID: 16566829
13.  Thallium ions can replace both sodium and potassium ions in the glutamate transporter EAAC1 
Biochemistry  2008;47(48):12923-12930.
The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl+ in the crystal structure, these sites were proposed to be Na+ binding sites. Therefore, we tested whether Tl+ has the ability to replace Na+ also in the mammalian transporters. Our data demonstrate that Tl+ can bind to EAAC1 with high affinity and mediate a host of different functions. Tl+ can functionally replace potassium when applied to the cytoplasm and support glutamate transport current. When applied extracellularly, Tl+ induces some behavior that mimics that of the Na+-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na+ in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na+ and Tl+ affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl+ is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K+, Tl+ is more efficient in mimicking K+ than Na+ when interacting with the mammalian protein.
doi:10.1021/bi8017174
PMCID: PMC2651767  PMID: 18986164
14.  Increased Expression of the Neuronal Glutamate Transporter (EAAT3/EAAC1) in Hippocampal and Neocortical Epilepsy 
Epilepsia  2002;43(3):211-218.
Summary
Purpose
To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy.
Methods
The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis.
Results
The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with nondysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue.
Conclusions
Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.
PMCID: PMC2441873  PMID: 11906504
Glutamate transporter; EAAT3/EAAC1; Epilepsy; Dentate; Dysplasia
15.  Expression of multiple glutamate transporter splice variants in the rodent testis 
Asian Journal of Andrology  2010;13(2):254-265.
Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of 𝒹-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.
doi:10.1038/aja.2010.99
PMCID: PMC3739219  PMID: 21170079
excitatory amino acid transporter; glutamate aspartate transporter; glutamate transporter 1; sperm; splice variant; testis; transporter
16.  The conserved histidine 295 does not contribute to proton cotransport by the glutamate transporter EAAC1 
Biochemistry  2005;44(9):3466-3476.
Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na+ ions and one proton. Previously, we suggested that the mechanism of H+ cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H+-binding network, possibly involving the conserved histidine 295 in the 6th transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady state electrophysiological analysis of the mutant transporters in order to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that can not be protonated, generates a fully functional transporter with transport kinetics that are close to the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH, and, thus, does not contribute to H+ cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues can not be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, non-transported proton to the amino acid side chain in position 295.
doi:10.1021/bi047812i
PMCID: PMC2430086  PMID: 15736956
Glutamate; excitatory amino acid transporter; EAAC1; glutamate transporter; mutagenesis; pre-steady-state kinetics
17.  Regional Distribution of Sodium-Dependent Excitatory Amino Acid Transporters in Rat Spinal Cord 
Background/Objective:
The excitatory amino acid transporters (EAATs), or sodium-dependent glutamate transporters, provide the primary mechanism for glutamate removal from the synaptic cleft. EAAT distribution has been determined in the rat brain, but it is only partially characterized in the spinal cord.
Methods:
The regional anatomic distribution of EAATs in spinal cord was assessed by radioligand autoradiography throughout cervical, thoracic, and lumbar cord levels in female Sprague-Dawley rats. EAAT subtype regional distribution was evaluated by inclusion of pharmacologic transport inhibitors in the autoradiography assays and by immunohistochemistry using subtype-specific polyclonal antibodies to rat GLT1 (EAAT2), GLAST (EAAT1), and EAAC1 (EAAT3) rat transporter subtypes.
Results:
[3H]-D-Aspartate binding was distributed throughout gray matter at the 3 spinal cord levels, with negligible binding in white matter. Inclusion of pharmacologic transport inhibitors indicates that the EAAT2/GLT1 subtype represents 21% to 40% of binding. Both EAAT1/GLAST and EAAT3/EAAC1 contributed the remainder of binding. Immunoreactivity to subtype-specific antibodies varied, depending on cord level, and was present in both gray and white matter. All 3 subtypes displayed prominent immunoreactivity in the dorsal horn. EAAT3/EAAC1 and to a lesser extent EAAT1/GLAST immunoreactivity also occurred in a punctate pattern in the ventral horn.
Conclusions:
The results indicate heterogeneity of EAAT distribution among spinal cord levels and regions. The presence of these transporters throughout rat spinal cord suggests the importance of their contributions to spinal cord function.
PMCID: PMC2031954  PMID: 17684893
Spinal cord; Glutamate plasma membrane proteins; Autoradiography; Immunohistochemistry; Amino acid transporters, Excitatory
18.  Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3) 
PLoS ONE  2014;9(10):e109245.
Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.
doi:10.1371/journal.pone.0109245
PMCID: PMC4183567  PMID: 25275463
19.  Isoforms of the neuronal glutamate transporter gene, SLC1A1/EAAC1, negatively modulate glutamate uptake: relevance to obsessive-compulsive disorder 
Translational Psychiatry  2013;3(5):e259-.
The SLC1A1 gene, which encodes the neuronal glutamate transporter, EAAC1, has consistently been implicated in obsessive-compulsive disorder (OCD) in genetic studies. Moreover, neuroimaging, biochemical and clinical studies support a role for glutamatergic dysfunction in OCD. Although SLC1A1 is an excellent candidate gene for OCD, little is known about its regulation at the genomic level. Here, we report the identification and characterization of three alternative SLC1A1/EAAC1 mRNAs: a transcript derived from an internal promoter, termed P2 to distinguish it from the transcript generated by the primary promoter (P1), and two alternatively spliced mRNAs: ex2skip, which is missing exon 2, and ex11skip, which is missing exon 11. All isoforms inhibit glutamate uptake from the full-length EAAC1 transporter. Ex2skip and ex11skip also display partial colocalization and interact with the full-length EAAC1 protein. The three isoforms are evolutionarily conserved between human and mouse, and are expressed in brain, kidney and lymphocytes under nonpathological conditions, suggesting that the isoforms are physiological regulators of EAAC1. Moreover, under specific conditions, all SLC1A1 transcripts were differentially expressed in lymphocytes derived from subjects with OCD compared with controls. These initial results reveal the complexity of SLC1A1 regulation and the potential clinical utility of profiling glutamatergic gene expression in OCD and other psychiatric disorders.
doi:10.1038/tp.2013.35
PMCID: PMC3669922  PMID: 23695234
EAAC1; EAAT3; internal promoter; obsessive-compulsive disorder; protein isoform; SLC1A1
20.  On the Mechanism of Proton Transport by the Neuronal Excitatory Amino Acid Carrier 1 
The Journal of General Physiology  2000;116(5):609-622.
Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na+, K+, and H+ concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)–expressing HEK293 cells with a rapid kinetic technique with a 100-μs time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the pre–steady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH− countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is ∼8. This value is shifted to ∼6.5 when the substrate binding site is exposed to the cytoplasm.
PMCID: PMC2229481  PMID: 11055990
glutamate transporter; patch-clamp; laser-pulse photolysis; rapid kinetics; reverse transport
21.  Simulated Whiplash Modulates Expression of the Glutamatergic System in the Spinal Cord Suggesting Spinal Plasticity Is Associated with Painful Dynamic Cervical Facet Loading 
Journal of Neurotrauma  2010;27(1):163-174.
Abstract
The cervical facet joint and its capsule have been reported to be injured during whiplash scenarios and are a common source of chronic neck pain from whiplash. Both the metabotropic glutamate receptor 5 (mGluR5) and the excitatory amino acid carrier 1 (EAAC1) have pivotal roles in chronic pain. In this study, spinal mGluR5 and EAAC1 were quantified following painful facet joint distraction in a rat model of facet-mediated painful loading and were evaluated for their correlation with the severity of capsule loading. Rats underwent either a dynamic C6/C7 joint distraction simulating loading experienced during whiplash (distraction; n = 12) or no distraction (sham; n = 6) to serve as control. The severity of capsular loading was quantified using strain metrics, and mechanical allodynia was assessed after surgery. Spinal cord tissue was harvested at day 7 and the expression of mGluR5 and EAAC1 were quantified using Western blot analysis. Mechanical allodynia following distraction was significantly (p < 0.001) higher than sham. Spinal expression of mGluR5 was also significantly (p < 0.05) greater following distraction relative to sham. However, spinal EAAC1 was significantly (p = 0.0003) reduced compared to sham. Further, spinal mGluR5 expression was significantly positively correlated to capsule strain (p < 0.02) and mechanical allodynia (p < 0.02). Spinal EAAC1 expression was significantly negatively related to one of the strain metrics (p < 0.003) and mechanical allodynia at day 7 (p = 0.03). These results suggest that the spinal glutamatergic system may potentiate the persistent behavioral hypersensitivity that is produced following dynamic whiplash-like joint loading; chronic whiplash pain may be alleviated by blocking mGluR5 expression and/or enhancing glutamate transport through the neuronal transporter EAAC1.
doi:10.1089/neu.2009.0999
PMCID: PMC2824231  PMID: 19772459
excitatory amino acid carrier 1; facet; glutamate; metabotropic glutamate receptor 5; pain
22.  Aberrant Rab11-Dependent Trafficking of the Neuronal Glutamate Transporter EAAC1 Causes Oxidative Stress and Cell Death in Huntington's Disease 
The Journal of Neuroscience  2010;30(13):4552-4561.
Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD140Q/140Q), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD140Q/140Q mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione.
doi:10.1523/JNEUROSCI.5865-09.2010
PMCID: PMC3842456  PMID: 20357106
23.  NEUTRALIZATION OF THE ASPARTIC ACID RESIDUE D367, BUT NOT D454, INHIBITS BINDING OF NA+ TO THE GLUTAMATE-FREE FORM AND CYCLING OF THE GLUTAMATE TRANSPORTER EAAC1 
The Journal of biological chemistry  2006;281(15):10263-10272.
Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na+ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, D367 and D454, in Na+ cotransport. In order to test the effect of charge neutralization mutations in these positions on Na+ binding to the glutamate-free transporter, we recorded the Na+-induced anion leak current to determine the Km of EAAC1 for Na+. For EAAC1WT, this Km was determined as 120 mM. When the negative charge of D367 was neutralized by mutagenesis to asparagine, Na+ activated the anion leak current with a Km of about 2 M, indicating dramatically impaired Na+ binding to the mutant transporter. In contrast, the Na+ affinity of EAAC1D454N was virtually unchanged compared to the wild type transporter (Km = 90 mM). The reduced occupancy of the Na+ binding site of EAAC1D367N resulted in a dramatic reduction in glutamate affinity (Km = 3.6 mM, 140 mM [Na+]), which could be partially overcome by increasing extracellular [Na+]. In addition to impairing Na+ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which D367, but not D454 is involved in coordinating the bound Na+ in the glutamate-free transporter form.
doi:10.1074/jbc.M510739200
PMCID: PMC2430067  PMID: 16478724
24.  Glutamate transporter expression and function in a striatal neuronal model of Huntington’s disease 
Neurochemistry international  2013;62(7):973-981.
Excitotoxicity may contribute to the pathogenesis of Huntington’s disease. High affinity Na+ dependent glutamate transporters, residing in the plasma membrane, clear glutamate from the extracellular space and are the primary means of prevention against excitotoxicity. Many reports suggest that Huntington’s disease is associated with a decrease in the expression and function of glutamate transporters. We studied the expression and function of these transporters in a cellular model of Huntington’s disease, STHdhQ111/Q111 and STHdhQ7/Q7 cells. We found that only GLT-1b and EAAC1 were expressed in these cell lines and only EAAC1 significantly contributed to the glutamate uptake. Surprisingly, there was an increase in Na+-dependent glutamate uptake in STHdhQ111/Q111 cells accompanied by an increase in surface expression of EAAC1 We studied the influence of the Akt pathway on EAAC1 mediated uptake, since EAAC1 surface expression is influenced by Akt and previous studies have shown increased Akt expression in STHdhQ111/Q111 cells. Glutamate uptake was inhibited by Akt pathway inhibitors in both the STHdhQ7/Q7 and the STHdhQ111/Q111 cell lines, and, in fact, we have found no difference in Akt activation between the two cell lines under our conditions of culture. Therefore a difference in Akt activation does not seem to explain the increase in EAAC1 mediated uptake in the STHdhQ111/Q111 cells.
doi:10.1016/j.neuint.2013.02.026
PMCID: PMC3653299  PMID: 23507328
Huntington’s disease; striatal; glutamate uptake; glutamate transporters; EAAC1
25.  Brimonidine prevents neurodegeneration in a mouse model of normal tension glaucoma 
Cell Death & Disease  2014;5(7):e1341-.
Glaucoma is one of the leading causes of irreversible blindness that is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino-acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs, and the loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP). Brimonidine (BMD) is an α2-adrenergic receptor agonist and it is commonly used in a form of eye drops to lower IOP in glaucoma patients. Recent studies have suggested that BMD has direct protective effects on RGCs involving IOP-independent mechanisms, but it is still controversial. In the present study, we examined the effects of BMD in EAAC1-deficient (KO) mice, an animal model of normal tension glaucoma. BMD caused a small decrease in IOP, but sequential in vivo retinal imaging and electrophysiological analysis revealed that treatment with BMD was highly effective for RGC protection in EAAC1 KO mice. BMD suppressed the phosphorylation of the N-methyl-D-aspartate receptor 2B (NR2B) subunit in RGCs in EAAC1 KO mice. Furthermore, in cultured Müller glia, BMD stimulated the production of several neurotrophic factors that enhance RGC survival. These results suggest that, in addition to lowering IOP, BMD prevents glaucomatous retinal degeneration by stimulating multiple pathways including glia–neuron interactions.
doi:10.1038/cddis.2014.306
PMCID: PMC4123097  PMID: 25032864

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